Peer Review #3 of "13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses (v0.2)"

Lactococcus lactissubsp.cremorisMG1363 is an important starter culture for dairy fermentation. During industrial fermentations,L. lactisis constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response ofL. lactisto several stresses has been described, the adaptation mechanisms at the level ofin vivofluxes have seldom been described. To gain insights into cellular metabolism,13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism ofL. lactiswhen subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability ofL. lactis’central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) inL. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering inL. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of howL. lactisregulates central metabolism under various conditions.

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Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-021-01526-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ying Li ◽

He Xian ◽

Ya Xu ◽

Yuan Zhu ◽

Zhijie Sun ◽

...

Keyword(s):

Pentose Phosphate Pathway ◽

Conversion Rate ◽

Metabolic Flux ◽

Flux Ratio ◽

Reducing Power ◽

Pentose Phosphate ◽

Fine Tuning ◽

High Yield ◽

Glycolytic Flux ◽

Acetyl Coa

Abstract Background Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and “bifid shunt”, to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). Result Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

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Bioreaction Network Topology and Metabolic Flux Ratio Analysis by Biosynthetic Fractional 13C Labeling and Two-Dimensional NMR Spectroscopy

Metabolic Engineering ◽

10.1006/mben.1999.0116 ◽

1999 ◽

Vol 1 (3) ◽

pp. 189-197 ◽

Cited By ~ 72

Author(s):

Thomas Szyperski ◽

Ralf W Glaser ◽

Michel Hochuli ◽

Jocelyne Fiaux ◽

Uwe Sauer ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Network Topology ◽

Metabolic Flux ◽

Flux Ratio ◽

Ratio Analysis ◽

Two Dimensional ◽

13C Labeling

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Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress

Molecular BioSystems ◽

10.1039/c2mb25285f ◽

2013 ◽

Vol 9 (1) ◽

pp. 121-132 ◽

Cited By ~ 9

Author(s):

Tie Shen ◽

Bin Rui ◽

Hong Zhou ◽

Ximing Zhang ◽

Yin Yi ◽

...

Keyword(s):

Oxidative Stress ◽

Metabolic Flux ◽

Metabolic Response ◽

Flux Ratio ◽

Ratio Analysis ◽

Multi Objective Optimization ◽

E Coli ◽

Multi Objective

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SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis

PLoS Computational Biology ◽

10.1371/journal.pcbi.1005109 ◽

2016 ◽

Vol 12 (9) ◽

pp. e1005109 ◽

Cited By ~ 22

Author(s):

Maria Kogadeeva ◽

Nicola Zamboni

Keyword(s):

Metabolic Flux ◽

Flux Ratio ◽

Ratio Analysis

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Methionine coordinates a hierarchically organized anabolic program enabling proliferation

10.1101/249367 ◽

2018 ◽

Cited By ~ 2

Author(s):

Adhish S. Walvekar ◽

Rajalakshmi Srinivasan ◽

Ritu Gupta ◽

Sunil Laxman

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Metabolic Flux ◽

Response Regulator ◽

Pentose Phosphate ◽

Dependent Manner ◽

Comparative Transcriptome ◽

Starvation Stress ◽

Underlying Basis ◽

Rate Limiting

AbstractMethionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here, we construct the organization of this methionine mediated anabolic program, using yeast. Combining comparative transcriptome analysis, biochemical and metabolic flux based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of three key regulatory nodes. These are: the pentose phosphate pathway coupled with reductive biosynthesis, and overall transamination capacity, including the synthesis of glutamate/glutamine. These provides the cofactors or substrates that enhance subsequent rate-limiting reactions in the synthesis of costly amino acids, and nucleotides, which are also induced in a methionine dependent manner. This thereby results in a biochemical cascade establishing an overall anabolic program. For this methionine mediated anabolic program leading to proliferation, cells co-opt a “starvation stress response” regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

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Methionine coordinates a hierarchically organized anabolic program enabling proliferation

Molecular Biology of the Cell ◽

10.1091/mbc.e18-08-0515 ◽

2018 ◽

Vol 29 (26) ◽

pp. 3183-3200 ◽

Cited By ~ 12

Author(s):

Adhish S. Walvekar ◽

Rajalakshmi Srinivasan ◽

Ritu Gupta ◽

Sunil Laxman

Keyword(s):

Amino Acid ◽

Metabolic Flux ◽

Response Regulator ◽

Pentose Phosphate ◽

Dependent Manner ◽

Comparative Transcriptome ◽

Starvation Stress ◽

Acid Biosynthesis ◽

Underlying Basis ◽

Rate Limiting

Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here we construct the organization of this methionine-mediated anabolic program using yeast. Combining comparative transcriptome analysis and biochemical and metabolic flux-based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH)-dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP)-dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine-mediated anabolic program to be sustained, cells co-opt a “starvation stress response” regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

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